ES2616435T3 - Cinacalcet and neuroblastic tumors - Google Patents

Abstract

The present invention is in the field of pediatric or developmental tumor therapy. Specifically, it refers to the use of the allosteric activator of the calcium sensor receptor, cinacalcet, for the preparation of a medicament for the treatment of neuroblastic tumors, in particular for the treatment of neuroblastomas, ganglioneuroblastomas and ganglioneuromas.

Description

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DESCRIPTION

Cinacalcet and neuroblastic tumors Field of the invention

The present invention belongs to the field of pediatric tumors or childhood development. Specifically, the invention relates to the allosteric activator of the calcium sensor receptor, cinacalcet, for use in the treatment of neuroblastic tumors.

Background of the invention

Childhood tumors are known as those that manifest primarily during pediatric age or that only manifest at this stage. For this reason they are sometimes also called pediatric tumors. In the context of the present invention the term childhood development tumor or pediatric tumor will be used interchangeably.

Within the group of childhood development tumors are neuroblastomas (NB), ganglioneuroblastomas (GNB) and ganglioneuromas (GN), which make up the group of neuroblastic tumors (TN). Neuroblastic tumors are the most frequent extracranial solid tumors of childhood and develop from cells of the neural crest already involved in the formation of the peripheral nervous system, so they are located in the adrenal medulla or in the nerve ganglia of the sympathetic paravertebral chain. The majority of localized NB, GNB and GN have excellent survival rates, but metastatic NBs, despite being subjected to multimodal intensive therapies, still have 40% survival rates.

Neuroblastic tumors are a group of tumors that are very heterogeneous in terms of climate, pathology, genetics and biology. The biological bases responsible for the chemical diversity of TNs are only partially known, but they are undoubtedly behind the different forms of tumor response to treatment protocols. Among them, the alterations of the ploidfa, the unbalanced translocations, the deletions or additions of recurrent chromosomal regions and the amplification of the MYCN oncogene stand out. These genetic-molecular alterations, although crucial for the biological behavior of TN, can hardly be modified therapeutically. Before they were described in more detail, it was known that the more differentiated TNs and with a greater proportion of Schwann stromal component (NB in differentiation, GNB and GN) were associated with a better prognosis than undifferentiated NBs. Some of the molecular pathways responsible for the processes of cell differentiation in TN have been discovered. These routes are of great therapeutic importance since they are pharmacologically modulable. Thus, for example, retinoic acid induces differentiation in NB cell lines and in patients' tumors, improving the overall survival of a subset of them. Some NBs are resistant to the action of this drug, but the lack of detailed knowledge of the molecular mechanisms responsible for the differentiation of TNs makes it difficult to design new differentiating agents that can benefit a greater number of patients.

The calcium sensor receptor (CASR) gene was cloned in 1993 from bovine parathyroid cells and is part of the C family of the G protein-coupled receptor superfamily (GpCR), together with eight glutamate receptors, two GABA receptors -B, some taste receptors and the GPRC6A amino acid sensor. This family of receptors detects signals of ions, amino acids and nutrients, among others, and transmits them to the intracellular environment. All of them share a similar structure that consists of a large amino-terminal extracellular domain, seven transmembrane helices and a carboxyl-terminal intracellular tail. In the case of CASR, its main function is to detect fluctuations of extracellular Ca2 + and consequently regulate the secretion of parathyroid hormone (PTH) in the parathyroid glands and calcitonin in the C cells of the thyroid, hormones responsible for normalizing calcemia. In the context of parathyroids, the activation of CASR by calcium results in a decrease in the regulation and production of parathyroid hormone (PTH), which in turn will decrease the plasma calcium concentration to keep it within a narrow range. (1.1-1.3 mM).

CASR expression has been found in different neoplasms with the peculiarity that very different functions are attributed, even contrasted, in different tumor contexts. Thus, for example, it has been described that the activation of CASR promotes proliferation and metastasis in prostate carcinoma, while the data seems to support that it participates in the differentiation processes in colon carcinoma.

There are direct CASR agonists (or type 1 agonists) and allosteric activators of CASR (or type 2 agonists). The former, such as Ca2 +, Mg +, Gd3 +, neomycin, L-amino acids, activate CASR by direct interaction with the extracellular domain thereof. On the other hand, allosteric activators do not activate the receptor by themselves but alter their three-dimensional structure, so that it becomes more sensitive to calcium stimulation. Cinacalcet is an allosteric activator of the calcium sensor receptor. Several CASR agonists are described in US Patent 5,688,938, such as NPS R-467 and NPS R-568, with NPS R-568 (also called R-568) being the most active. Also, US Patent 6,296,833 describes the use of calcium agonists or antagonists for the treatment of cancer. The present application describes the use of cinacalcet for the preparation of a medicine for

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treatment of neuroblastic tumors. Surprisingly, the use of cinacalcet results in important advantages since it is capable of inducing apoptosis of tumor cells at a lower concentration than other CASR agonists.

Object of the invention

The present invention relates to cinacalcet for use in the treatment of neuroblastic tumors, in particular for the treatment of neuroblastomas, ganglioneuroblastomas and ganglioneuromas.

Brief description of the figures

Figure 1A shows the morphology of SK-N-LP cells with (lower panels) or without (upper panels) CASR overexpression after 16 hours subjected to serum deprivation and subsequent exposure to 3 mM CaCl2 for 24 and 48 hours. Figure 1B shows an analysis of cell viability in the SK-N-LP cell line, with overexpression of CASR (black bar) or without overexpression of CASR (vada bar), after 16 hours of serum deprivation and subsequent exposure to CaCl2 0 , 5 mM, 1.4 mM or 3 mM for 48 hours. Figure 1C shows a Western blot analysis of caspase 3 activation and PARP cleavage in native SK-N-LP cells (wild type, WT) and SK-N-LP cells with CASR overexpression (CASR) after being exposed for 24 hours (first two lanes) and 48 hours (last two lanes) to the in vitro model described in section A.

Figure 2 shows the cell viability of native neuroblastoma cell lines treated in vitro with cinacalcet or with NPS R-568 for 72 hours. The percentage of viability with respect to the control versus concentration (Conc.) In pM of cinacalcet (circle) or R-568 (square) is represented. The analyzed cell lines are the following: LAN-1 (Fig. 2A), SK-N-LP (Fig. 2B), SK-N-JD (Fig. 2C), SK-N-BE (2) c (Fig 2D), LA1-5s (Fig. 2E), LA1-55n (Fig. 2F) and SK-N-AS (Fig. 2G).

Figure 3 shows the morphological examination of xenoimplants generated from the cell lines LAN-1 (Fig. 3A, 3C, 3E) and SK-N-LP (Fig. 3B, 3D, 3F) treated with cinacalcet (Fig. 3C -3F) or not (Fig. 3A, 3B). Specimens are shown in which cinacalcet produced mostly cell death (Fig. 3C, 3D) or cytodifferentiation (Fig. 3E, 3F).

Detailed description of the invention

In one aspect, the present invention relates to cinacalcet for use in the treatment of neuroblastic tumors. In a particular embodiment, it refers to cinacalcet for use in the treatment of neuroblastomas. In another particular embodiment, it refers to cinacalcet for use in the treatment of ganglioneuroblastomas. In another particular embodiment, it refers to cinacalcet for use in the treatment of ganglioneuromas.

As mentioned in the background section, US Patent 5,688,938 describes NPS R-568 as the most active CASR agonist compound. In this regard, comparative trials of the effects of the use of cinacalcet and R-568 in the treatment of neuroblastic tumors are carried out in the present invention. Surprisingly, cinacalcet has a number of advantages over NPS R-568 from a pharmacological and pharmacodynamic point of view. One of the most important and surprising effects of cinacalcet is the induction of apoptosis at significantly lower concentrations than the NPS R-568 (Figure 2, Table 1). This implies important advantages of cinacalcet over other CASR agonists as it allows an effective treatment with the administration of a lower dose of medication which generally implies a lower risk of side effects or toxicity and greater tolerance to treatment, in addition to facilitating the administration and administration guideline. These advantages are especially relevant when a drug must be administered steadily over time, such as cinacalcet administered for the treatment of neuroblastic tumors. These tumors have frequent relapses, which makes it foreseeable that this treatment, as well as others used in the context of minimal residual disease to prevent relapse, should be administered continuously for a long period of time. Likewise, it is possible that if a tumor has responded adequately to the initial treatment with cinacalcet, it may benefit from the reuse of this drug if it reappears (after the initial remission), a strategy that has been shown to be useful with various treatments in oncology (rechallenging) .

Examples

Below are some concrete examples of realization of the invention that serve to illustrate the invention.

Example 1. Analysis of the effects promoted in vitro by the activation of CASR by calcium in neuroblastoma cell lines with or without CASR overexpression.

Native SK-N-LP cells, stably transfected with pCMV-GFP or with pCMV-CASR-GFP (1x106) in RPMI 10% FBS were seeded on P100 plates. The next day, the medium was replaced by a serum-free (calcium-free DMEM supplemented with 0.2% bovine albumine, 4 mM L-glutamine and 0.5 mM CaCl2). This was maintained for 16 hours, before replacing it with the same medium supplemented or not with CaCl2 up to 3 mM. It was thus observed that cells with overexpression of CASR suffer cell death in this model when they were exposed to CaCl2

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3 mM (Figure 1A, bottom panel) but not at 0.5 mM (data not shown). This phenomenon, which began about 6 hours after treatment with calcium and was massive after 48 hours, was barely noticeable in native cell lines or transfected with pCMV-GFP (Figure 1A, upper panel). In the latter, signs of cell death were detected, but the cells continued to proliferate until they reached confluence, while the cells with overexpression of CASR disappeared completely within 5 days.

To quantify the decrease in cell viability, the same model was made in 24-well plates in the lines and clones mentioned above (Figure 1B). At 48 hours, the percentage of viable cells was quantified using the Promega CellTiter 96 (r) Aqueous One Solution Cell Proliferation Assay (MTS) system. It was thus observed that the cell viability of the cells with overexpression of CASR (black bar, Figure 1B) decreases significantly more than that of the native SK-N-LP cell line (bar vada, Figure 1B).

In order to determine whether the process of cell death is subject to apoptosis, total proteins were extracted from native SK-N-LP (wild-type, WT) cells and with overexpression of CASR (CASR) at 24 and 48 hours after Exposure to this model. The proteins were subjected to SDS-PAGE gel electrophoresis, transferred to nitrocellulose membrane and incubated with specific antibodies to detect activated caspase 3 and c-PARP (Cell Signaling). Appropriate peroxidase-labeled secondary antibodies were used and development was carried out with the Amersham ECL system. Thus, it was revealed that cell death induced by activation of CASR by calcium is accompanied by activation of caspase 3 and cleavage of PARP (Figure 1C), which is compatible with cell death by apoptosis.

Example 2. Analysis of the effects on cell viability promoted in vitro by cinacalcet and NPS R-568 in seven neuroblastoma cell lines.

5x103 cells of each cell line (LAN-1, SK-N-BE (2) c, LA1-55n, LA1-5s, SK-N-LP, SK-N-JD, SK-N-AS) were seeded in 96-well plates (6 replicates per condition). The following day, the initial medium (RPMI 10% FBS) was removed and replaced by the same medium that contains various concentrations (100 | jM, 65 | jM, 30 | jM, 25 | jM, 20 | jM, 15 | jM , 10 jiM, 5 jiM, 3 jiM, 1 jiM, 0.3 jiM) of cinacalcet (Selleck Biochemicals) or NPS R-568 (Tocris) or equivalent amounts of DMSO (Sigma-Aldrich). At 72 hours, cell viability was assessed by quantifying the percentage of viable cells using the Promega CellTiter 96 (r) Aqueous One Solution Cell Proliferation Assay (MTS) system. It was observed that both drugs induce a decrease in cell viability in the 7 cell lines examined (Figure 2), and that in 5 of them the IC50 (drug concentration that causes the death of 50% of the cells with respect to the control ) of cinacalcet was lower than that of NPS R-568 in a statistically significant way (Table 1). In the case of the SK-N-AS and LA1-55n cell lines, the difference in the effect promoted by both drugs was not statistically significant (Table 1).

Table 1. IC50 values obtained by treating seven native cell lines of neuroblastoma with cinacalcet or NPS R-568.

 Cell phone

 Cinacalcet NPS R-568 P value

 LAN-1

 17.19 24.81 <0.0001

 SK-N-LP

 10.31 22.38 <0.0001

 SK-N-JD

 10.92 25.82 <0.0001

 SK-N-BE (2) C

 10.69 16.65 <0.0001

 LA1 -5s

 16.73 34.5 <0.0001

 LA1-55n

 10.52 11.42 not significant

 SK-N-AS

 10.26 15.14 not significant

Example 3. Analysis of the effects of cinacalcet treatment in an in vivo neuroblastoma model.

Xenoimplants of the LAN-1 and SK-N-LP cell lines were generated for which almuots of 10x106 cells were inoculated from said cell lines subcutaneously in 4-6 weeks atomic nu / nu female mice (Charles Rivers). From day 12, when the tumor in formation was already measurable with king's foot, the treatment of 6 days per week with 100 mg / kg cinacalcet was started orally (Figures 3C-3F) and with the vehicle as a negative control (Figures 3A and 3B). Tumor volume was measured every 2-3 days. At 40 days, the mice were sacrificed. Each tumor was weighed before dividing it into two halves: one freezing in liquid nitrogen for expression analysis and the other was fixed and embedded in paraffin for morphological examination and immunohistochemical analysis. In the morphological analysis of the xenoimplants of both cell lines treated with

cinacalcet, spurs were observed in which there are large areas with necrosis and / or apoptosis (Figures 3C and 3D) and / or morphological signs of differentiation in the cells that were viable (Figures 3E and 3F).

Claims (1)

1) Cinacalcet for use in the treatment of neuroblastic tumors. 2) Cinacalcet for use according to claim 1, in which the neurobastic tumor is a neuroblastoma. 3) Cinacalcet for use according to claim 1, wherein the neurobastic tumor is a ganglioneuroblastoma. 4) Cinacalcet for use according to claim 1, wherein the neurobastic tumor is a ganglioneuroma.